For example, in molecular biology, hybridization means a phenomenon in which nucleic acids hydrogen-bond to each other through complementary base pairing. In other words, if the nucleotide sequence of a nucleic acid molecule to be measured is known, a nucleic acid molecule having a nucleotide sequence complementary to the nucleotide sequence can be used to detect the nucleic acid molecule to be measured. More specifically, this is a method which involves reacting a fluorescence-labeled nucleic acid to be measured with a solid phase on which a nucleic acid probe having a nucleotide sequence complementary to the nucleic acid molecule to be measured is immobilized, washing/removing the unreacted nucleic acid molecules, and measuring the activity of the labeled substance bound to the solid phase. The hybridization method can detect a nucleic acid molecule to be measured by accurately recognizing the DNA sequence.
To accurately detect a nucleic acid molecule to be measured in the hybridization, it is important for a nucleic acid probe to accurately recognize the nucleic acid molecule. Hence, in conducting hybridization, a method involving properly regulating the salt concentration and reaction temperature of a reaction solution or a method involving using a blocking agent for suppressing non-specific hybridization of a nucleic acid probe with nucleic acid molecules other than that to be measured have conventionally been used. Known examples of the blocking agent include nucleic acid components not having nucleotide sequences complementary to a nucleic acid molecule to be measured and a nucleic acid probe, such as salmon sperm DNA and yeast tRNA, surfactants, such as SDS (sodium dodecyl sulfate) and N-lauroyl sarcosine (N-LS), and proteins, such as bovine serum albumin (BSA) and casein.
However, when nucleic acid molecules not to be measured are abundantly present, there are problems that the blocking agents consisting of nucleic acid components exhibit insufficient blocking effects and the surfactants and the proteins have weak blocking effects because they cannot exactly recognize nucleotide sequences.
Patent Literature 1 discloses a method which involves using a blocker probe hybridizing with a unique sequence in a nucleic acid molecule to be measured and specifically hybridizing with a capture sequence probe containing a nucleic acid sequence captured on a solid phase. In the method described in Patent Literature 1, the blocker probe is added to a reaction solution after the hybridization of the capture sequence probe to the nucleic acid molecule to be measured to prevent the unhybridized capture sequence probe from hybridizing with a crossreactive nucleic acid sequence present in the nucleic acid molecule to be measured, thereby enabling the enhancement of detection specificity.
In addition, Patent Literature 2 discloses the use of an oligonucleotide containing a modified nucleotide, such as a locked nucleic acid (LNA), as a blocking agent when a nucleic acid molecule to be measured is detected using a microarray.
Furthermore, Patent Literature 3 discloses a method which involves detecting a nucleic acid molecule to be measured with a nucleic acid probe using a 5′-terminal block nucleic acid hybridizing with 5′-end side to a nucleotide to be detected in a nucleic acid molecule to be measured and a 3′-terminal block nucleic acid hybridizing with 3′-end side to the nucleotide to be detected. The method in Patent Literature 3 is considered to have a high nucleotide sequence specificity in hybridization of the probe nucleic acid with the target nucleic acid, enabling the enhancement of the efficiency and specificity of the typing of SNP required to detect the difference of only one nucleotide in a nucleotide sequence with high accuracy and the detection and separation of a nucleic acid having a particular nucleotide sequence.